PROJECT SUMMARY Heart failure with preserved ejection fraction (HFpEF) is the most common form of heart failure in patients over 65 years of age. Current therapeutic regimens do not effectively treat HFpEF; therefore, it is critical to understand cellular mechanisms that cause this disease. Importantly, as women age, they have a higher risk of developing HFpEF than men. It is not known what leads to this increased risk in females. Since a hallmark feature of HFpEF is impaired relaxation, an important step in elucidating why women are at higher risk of developing HFpEF is to understand how factors that are integral to relaxation differ between males and females and how they change with age. This proposal seeks to elucidate how relaxation is regulated in males and females in normal cardiac aging at the most basic level of cardiomyocyte function, the sarcomere. By studying mechanical parameters of small bundles of sarcomeres, myofibrils, key differences in relaxation can be defined and mechanisms that contribute to differences can be identified. Preliminary studies demonstrate relaxation is prolonged in myofibrils isolated from hearts of female donors over 60 years of age compared to myofibrils isolated from hearts of female donors between 20 and 40 years of age. Additionally, there are also sex-differences in myofibril relaxation. Further preliminary studies also suggest histone deacetylase 8 (HDAC8) may play a role in regulation of relaxation through acetylation of sarcomeric proteins. This proposal will define sex differences in myofibril acetylation and relaxation in non-failing human hearts from young adults (20-40 years of age) and older adults (> 60 years of age) (Aim 1). Since myofibril relaxation changes observed in humans are recapitulated in adult (5 months of age) and older (20 months of age) male and female mice, in vitro and in vivo mouse models will be used to interrogate changes in myofibril relaxation in aging and with manipulation of HDAC8 (Aim 2). Identifying how aging affects myofibril relaxation in males and females will provide valuable insight into mechanisms that contribute to the development of HFpEF. This proposal is designed to expand Dr. Woulfe's research to study cardiac aging. Dr. Woulfe's overall career goal is to study sex-differences in myofibril mechanics across the lifespan and the training outlined in this proposal will allow her to develop key skills and experience necessary to study cardiac aging. Dr. Woulfe has established a mentorship team with expertise in aging, sex differences, and molecular biology of adenovirus generation and HDAC biology. Together, this team of uniquely qualified mentors and Dr. Woulfe have developed a custom training plan tailored to provide the skills and knowledge necessary to ensure appropriate research design and practices in a new area of research.